Genipin as a sporicidal agent for the treatment of cortical bone allografts

Introduction: Microbial bioburden in bone allografts can be reduced by gamma radiation; however, the radiation compromises collagen and increases the risk of graft failure. Genipin is an agent that may reduce bioburden by chemical crosslinking without causing mechanical detriment or cytotoxicity.

Methods: To evaluate genipin's ability to penetrate cortical bone while maintaining sporicidal activity, Bacillus subtilis spore strips were isolated between slices of bovine femoral cortical bone and immersed in genipin solutions for up to one week; spore viability was assessed with media-based assays. The mechanical effects of genipin treatment were assessed by performing three-point bending tests on genipin-treated cortical beams. Cytotoxicity studies were conducted by evaluating the adhesion and proliferation of murine MC3T3-E1 (P21) preosteoblasts on cortical bone slices which were treated with genipin and rinsed to different extents.

Results: Genipin successfully penetrated cortical bone slices and sterilized B. subtilis populations after 48 hours (p>0.05) and one week (p<0.05). Genipin-treated cortical beams demonstrated dose-dependent increases in yield strain (p=0.02) and resilience (p<0.01), whereas other mechanical properties were not affected by genipin treatment. Seeding cells onto inadequately rinsed genipin-treated bones proved cytotoxic. However, with adequate post-treatment rinsing of the residual genipin, cell adhesion and proliferation was comparable to phosphate-buffered saline-treated controls (no genipin).

Conclusions: Genipin solutions can sterilize bacterial spore populations entrapped within the continuum of bone tissue while preserving mechanical properties of bone and allowing cell adhesion and proliferation. Provided that antimicrobial effects seen with bacterial spores extend broadly to all microbial and viral species, genipin holds strong potential for bone allograft sterilization.